The invention relates to an arrangement and a method for generating and depositing a stream of fluid segments which are respectively separated by a intermediary medium that cannot be mixed with the fluid segments. The invention also relates to an apparatus, comprising such an arrangement, which is attached to a device for storing liquids (microtiter plate) and to the use of such an arrangement.
The question of how to adapt micro-fluidic arrangements, presently developed for use on a laboratory scale, to the microtiter plates that conform to the industrial standard is one of the biggest problems with respect to utilizing micro-fluidic arrangements in industrial-scale applications.
Microtiter plates, having measurements of 127.76 mm×85.48 mm×14.35 mm, are approximately postcard-sized components, made primarily of polymer, which contain a plurality of small depressions or wells. In the standardized form according to ANSI/SBS 1-2004, they have proven themselves as standard equipment for a plurality of uses in the life sciences and therefore represent the preferred standard systems for storing a large number of identical or different samples in liquid form in substance libraries, for example pharmaceuticals or DNA sequences. Microtiter plates are used as parallel reaction vessels in the field of combinatorial chemistry or for active agent screening. A plurality of chemical experiments can thus be realized simultaneously in the separate wells of a microtiter plate. Depending on the size of the selected plate, 6, 12, 24, 48, 96, 384 or 1536 wells serve as parallel reaction vessels. In present times, many processes realized in the pharmaceutical chemistry are no longer conceivable without the use of microtiter plates.
An extremely high throughput can be reached when using microtiter plates for screening, synthesis or analyzing processes along with standardized robotics. Since the goal of many industrial processes is the optimization of a high throughput, the use of the microtiter plate as a central component of this technology is of critical importance.
The main disadvantages of existing technologies using microtiter plates is the fact that most processes take place under ambient conditions and that the microtiter plate has a high interaction surface between the sample contained therein and the environment as a result of its opening cross section. Possible consequences include sample contamination and a danger of sample loss through evaporation. The use of robotics limits the operating speed since bulk amounts must be moved with the aid of pivoting arms or transport systems. The generated inertial forces acting upon the mechanical system during acceleration or deceleration must be compensated for or limited, to avoid an incorrect positioning or damage to the components.
Micro-fluidics represents an alternative way of achieving high throughputs with comparable processes since it permits the moving, mixing, reacting, separating and analyzing of small amounts of liquid. This takes place either in stationary devices such as micro-fluidic channels, or in micro-reaction chambers, or in active structures such as micro pumps or micro valves, thus avoiding the problem of moving bulk materials. Micro-fluidics can furthermore also integrate processing steps in addition to the pure extraction. In contrast, robotic systems frequently can be utilized only for the extraction of liquids from the microtiter plate.
Differently designed robotic systems are available from numerous manufacturers. Complete systems are often described which do not necessarily have a micro-fluidic periphery, for example as disclosed in European patent document EP 2052776 A1. The extraction process that is frequently used applies a vacuum pressure for suctioning a defined amount of liquid into a single-use tip of a vacuum pipette. Robotics permits the parallelization of this extraction process through a parallel activation of several suctioning devices. Units of the size of microtiter plates are available for the parallel extraction.
These devices have the disadvantage that they only permit the extraction of a sample, meaning the problem of connecting to the micro-fluidic devices is not solved. A further problem is that the sample is exposed completely to ambient air which can lead to contamination and/or evaporation and the resulting sample loss.
A significant disadvantage of the known micro-fluidic systems is that developments are often restricted to the use of only a single component. For example, a detector was optimized for detecting specific analyst or a pump for achieving fixed performance goals, or a chemical reaction was realized in a specific channel that is produced from a special type of material.
Equivalents to the macroscopic industrial processes were developed over the years, which in many cases made it possible to achieve comparable results with significantly reduced sample volumes. It is thus conceivable to have a reduction of expensive educts as a result of the miniaturization along with a scaling of the processes to achieve high parallelism. However, to date only a few micro-fluidic systems have actually found their way out of the laboratory to be used on an industrial scale. One of the most important reasons for this is the lack of adaptability to existing industrial systems.
Solutions have been described from time to time for connecting micro-fluidic systems to microtiter plates.
U.S. Patent Application Publication No. 2003/224531 A1 discloses a microtiter plate in which parallel reactions can take place and from which the formed products can be released with the aid of an electro-spray to a mass spectrometer. However, liquids cannot be extracted with this arrangement and, in addition, no standard microtiter plate is used but a micro-fluidic system which resembles the shape of a microtiter plate.
International patent publication WO 1/73396 A1 shows a micro-fluidic arrangement which is directly connected to a well via a capillary element that continuously pulls fluid from the well of a microtiter plate and into a micro-fluidic system. This arrangement has the disadvantage that the liquid flow and the amount of liquid to be extracted can be adjusted only with limitations and that the liquid is in contact with the environment. The arrangement is furthermore difficult to set up and control. In particular, it is difficult to extract liquid from different wells of a microtiter plate since the system is designed to be completely passive and the capillary effect cannot be easily stopped. A reverse depositing of the liquid into the well is furthermore also not possible since the direction of capillarity cannot be reversed.
U.S. Patent Application Publication No. 2005/047962 A1 describes a device capable of dispensing defined drops from a nozzle into a microtiter plate, in a manner similar to an inkjet printing head. The process is realized by applying pressure to a membrane which causes a drop to shoot out of the nozzle. The device has the disadvantage that it is suitable only for dispensing a droplet into a microtiter plate. In order to remove a droplet from the microtiter plate, it is necessary for the microtiter plate to be embodied as a nozzle plate.
U.S. Pat. No. 6,274,091 B1 describes a device using a vacuum to remove liquids from specially designed microtiter plates. For this, a microtiter plate is provided on the underside with an outlet, e.g. in the form of a nozzle, for suctioning the content out of the well. Alternatively, the well itself can be closed off and evacuated, causing the liquid to evaporate. In both cases, the volume amount to be extracted cannot be adjusted, so that the complete well is emptied in many cases. This device is furthermore not capable of dispensing liquid into a well.
A fluid exchanger is described in German patent document DE 10 2007 032 951 A1 and the article by B. E. Rapp, L. Carneiro, K. Lange and M. Rapp: “An indirect microfluidic flow injection analysis (FIA) system allowing diffusion free pumping of liquids by using tetradecane as intermediary liquid,” published in Lab Chip, 9, pp 354-356, 2009, in which one liquid can be exchanged against another liquid, provided the two liquids cannot be mixed chemically. This condition can be met, for example, by using a watery phase and oil as an intermediary medium. If both liquids are filled into a vessel which is sealed airtight, a stable two-phase boundary adjusts, wherein the liquid with the lower density is at the top. By inserting two access lines into the vessel, wherein each access line is respectively in contact with one of the two phases, one liquid can be exchanged for another liquid. If an intermediary medium is fed into the associated phase via the intake connected thereto, the same volume of the watery phase flows out of the vessel through the other access line, thus exchanging the two liquids.